L-Glutamic acid decarboxylase (GAD) catalyzes the synthesis of .gamma.-aminobutyric acid (GABA), which is widely accepted as the major inhibitory neurotransmitter in the mammalian brain. One isoform of GAD has been identified as a 65 kD beta cell autoantigen in the pancreatic islets of Langerhans. Type 1 (insulin-dependent) Diabetes mellitus (IDDM) is an autoimmune disease that leads to the destruction of the pancreatic beta-cells. Development of IDDM has been associated with the presence of autoantibodies to the 65 kD GAD enzyme.
Naturally occurring GAD65 is difficult to isolate from pancreatic islet cells in meaningful quantity and purity, and purified GAD has been isolated from COS cells in only trace amounts. Tuomi et al., Diabetes 42:359-362 (1993). The cloning of human islet cell GAD.sub.65 makes it theoretically possible to obtain recombinant protein useful as antigen in quantitative assays for measuring GAD autoantibodies in IDDM susceptible individuals. GAD65 has been expressed in bacterial (e.g., Atkinson et al., Lancet 339:458-459 (1992)), mammalian (e.g., Hagopian et al., Diabetes 42:1-3 (1993)), and insect cells (e.g., Christgau et al., J. Cell. Biol. 118:309-320 (1992)). These strategies provided partially purified material for the detection of autoantibodies on a small scale. However, they were not fully successful as sources of purified GAD, either because the level of expression was low, making purification difficult, enzymatic activity was reduced, or because the GAD65 was expressed as a fusion protein which might have altered the immunoreactivity of the GAD65 portion of the molecule. For example, the amount of GAD65 expressed from BHK cells was approximately 600 .mu.g/liter (Moody et al., Diabetologia 38:14-23 (1995), which made purification by affinity chromatography essential.
GAD65 is a complex molecule, containing 15 cysteine residues and two palmitoylated sites (Shi et al., J. Cell Biol. 124:927-934 (1994)). In solution GAD65 aggregates rapidly to form both covalent and non-covalently bound oligomers having reduced enzymatic activity and reduced ability to react with antibodies. These characteristics make the purification of recombinant GAD65 difficult. The material is often purified at high concentration under which conditions its inherent tendency to irreversibly aggregate leads to the formation of unusable precipitates.
The production of large quantities of GAD65 is sought as a source of antigen for development of immunoassays and potentially for use in screening and monitoring large numbers of individuals for susceptibility to Type 1 diabetes. However, large quantities of recombinant GAD65 are of little use if the biological characteristics of the molecule, such as antigenic properties and enzymatic activity, are impaired.
More recently GAD65 has been expressed in insect cells, such as Spodoptera frugiperda (Sf9) cells, using baculovirus vectors. Moody et al., supra; Mauch et al., J. Biochem. 113:699-704 (1993); and Christgau et al., J. Cell. Biol. 118:309-320 (1992). The recombinant GAD65 was reportedly obtained in large quantities from the insect cells (up to 50 mg/L) and could be purified up to 95% purity while retaining significant enzymatic and antigenic reactivity. Moody et al., ibid. However, insect cell expression systems suffer from a number of disadvantages when used for protein expression on an industrial scale. For example, the insect cells are difficult to manipulate in quantities needed to cells (1) are expensive to culture; (2) require infection with baculovirus for heterologous protein expression, making them unsuitable for continuous production methods; (3) produce poorly reproducible results, making them difficult to rigorously validate; and (4) have a low overall productivity rate.
What is needed in the art is a means for convenient expression of very large amounts of biologically active recombinant GAD65. The protein preparations isolated from the expression system should be readily purified to relative homogeneity, while retaining a high level of enzymatic and antigenic activity. Quite surprisingly, the present invention fulfills these and other related needs.